Apigenin and its combination with Vorinostat induces apoptotic-mediated cell death in TNBC by modulating the epigenetic and apoptotic regulators and related miRNAs.

Triple-negative breast cancer (TNBC) is a metastatic disease and a formidable treatment challenge as it does not respond to existing therapies. Epigenetic regulators play a crucial role in the progression and metastasis by modulating the expression of anti-apoptotic, pro-apoptotic markers and related miRNAs in TNBC cells. We have investigated the anti-TNBC potential of dietary flavonoid 'Apigenin' and its combination with Vorinostat on MDA-MB-231 cells. At Apigenin generated ROS, inhibited cell migration, arrested the cell cycle at subG0/G1 phases, and induced apoptotic-mediated cell death. Apigenin reduced the expression of the class-I HDACs at the transcriptomic and proteomic levels. In the immunoblotting study, Apigenin has upregulated pro-apoptotic markers and downregulated anti-apoptotic proteins. Apigenin inhibited the enzymatic activity of HDAC/DNMT and increased HAT activity. Apigenin has manifested its effect on miRNA expression by upregulating the tumor-suppressor miR-200b and downregulation oncomiR-21. Combination study reduced the growth of TNBC cells synergistically by modulating the expression of epigenetic and apoptotic regulators. Molecular docking and MD simulations explored the mechanism of catalytic inhibition of HDAC1 and HDAC3 and supported the in-vitro studies. The overall studies demonstrated an anti-TNBC potential of Apigenin and may help to design an effective strategy to treat metastatic phenotype of TNBC.

Targeting AKT2 in MDA-MB-231 Cells by Pyrazole Hybrids: Structural, Biological and Molecular Docking Studies.

Pyrazolic hybrids appended with naphthalene, p-chlorobenzene, o-phenol and toluene have been synthesized using Claisen Schmidt condensation reaction of 1-benzyl-3,5-dimethyl-1H-pyrazole-4-carbaldehyde. All compounds were characterized by various spectroscopic techniques. Compound (E)-3-(1-benzyl-3,5-dimethyl-1H-pyrazol-4-yl)-1-(4-chlorophenyl)prop-2-en-1-one crystallizes in monoclinic crystal system with C2/c space group. These synthesized compounds were tested for cytotoxic activity and among these compounds 4b and 5a shows prominent cytotoxic activity against triple-negative breast cancer (TNBC) cells MDA-MB-231 with IC50 values 47.72 µM and 24.25 µM, respectively. Distinguishing morphological changes were noticed in MDA-MB-231 cells treated with pyrazole hybrids contributing to apoptosis action. To get more insight into cytotoxic activity, in silico molecular docking of these compounds were performed and the results suggested that (E)-3-(1-benzyl-3,5-dimethyl-1H-pyrazol-4-yl)-1-(p-tolyl)prop-2-en-1-one and 1-(1'-benzyl-5-(4-chlorophenyl)-3',5'-dimethyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-yl)ethan-1-one binds to the prominent domain of Akt2 indicating their potential ability as Akt2 inhibitor. Moreover, from in silico ADME studies clearly demonstrated that these compounds may be regarded as a drug candidate for sub-lingual absorption based on log p values (2.157-4.924). These compounds also show promising antitubercular activity. The overall results suggest that pyrazolic hybrids with substitution at less sterically hindered positions have appealing potent cytotoxic activity and antituberculosis activity due to which they may act as multidrug candidate.

Repurposing of neprilysin inhibitor 'sacubitrilat' as an anti-cancer drug by modulating epigenetic and apoptotic regulators.

Modifications in the epigenetic landscape have been considered a hallmark of cancer. Histone deacetylation is one of the crucial epigenetic modulations associated with the aggressive progression of various cancer subtypes. Herein, we have repurposed the neprilysin inhibitor sacubitrilat as a potent anticancer agent using in-silico protein-ligand interaction profiler (PLIP) analysis, molecular docking, and in vitro studies. The screening of PLIP profiles between vorinostat/panobinostat and HDACs/LTA4H followed by molecular docking resulted in five (Sacubitrilat, B65, BDS, BIR, and NPV) FDA-approved, experimental and investigational drugs. Sacubitrilat has demonstrated promising anticancer activity against colorectal cancer (SW-480) and triple-negative breast cancer (MDA-MB-231) cells, with IC50 values of 14.07 µg/mL and 23.02 µg/mL, respectively. FACS analysis revealed that sacubitrilat arrests the cell cycle at the G0/G1 phase and induces apoptotic-mediated cell death in SW-480 cells. In addition, sacubitrilat inhibited HDAC isoforms at the transcriptomic level by 0.7-0.9 fold and at the proteomic level by 0.5-0.6 fold as compared to the control. Sacubitrilat increased the protein expression of tumor-suppressor (p53) and pro-apoptotic makers (Bax and Bid) by 0.2-2.5 fold while decreasing the expression of anti-apoptotic Bcl2 and Nrf2 proteins by 0.2-0.5 fold with respect to control. The observed cleaved PARP product indicates that sacubitrilat induces apoptotic-mediated cell death. This study may pave the way to identify the anticancer potential of sacubitrilat and can be explored in human clinical trials.

Prosopis juliflora (Sw.) DC phytochemicals induce apoptosis and inhibit cell proliferation signaling pathways, EMT, migration, invasion, angiogenesis and stem cell markers in melanoma cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Prosopis juliflora (Sw.), DC is a xerophytic plant species that extensively grow in Asia, Africa, Australia, and Brazil. From ancient time P. juliflora is being utilized in various folk remedies for example in wound healing, fever, inflammation, measles, excrescences, diarrhea and dysentery. Traditionally, gum, paste, and smoke obtained from the leaves and pods are applied for anticancer, antidiabetic, anti-inflammatory, and antimicrobial purposes. AIM OF THE STUDY: Our previous studies have demonstrated the promising potential of Prosopis Juliflora leaves methanol extract (PJLME) against breast cancer, and suggested its possible integration as a complementary medicine for the effective management of breast cancer. However, evidence against how PJLME mechanistically target the cancer proliferative pathways and other targets is poorly understood. The basic aim of the present study was to understand the anti-melanoma potential of PJLME against B16f10 cells with possible mechanisms of action. MATERIALS AND METHODS: MTT assay was used to determine cell viability. Wound and transwell migration assay was performed to check migration potential of cells after PJLME treatment, while clonogenic assay was carried out to understand its colony inhibition actvity. Flow cytometry was used to perform annexin V/PI assay (apoptosis assay), ROS assay, cell cycle analysis. In-vitro angiogenesis assay was performed to check formation of capillary like vascular structure after PJLME treatment. Apoptotic genes, signaling pathways markers, EMT markers and stem cell markers were determined by western blotting. In-vivo BALB/C mice xenograft model study was performed to check the effect of PJLME on in-vivo melanoma tumor growth. RESULTS: The experimental outcome of the present study has clearly demonstrated the inhibition of growth, migration, invasion, colony formation and apoptosis inducing potential of PJLME against mouse melanoma cancer cells. Treatment of B16F10 melanoma cells with PJLME resulted in arrest of cell cycle at G0/G1 phase. Annexin V-FITC/PI assay confirmed the apoptosis inducing potential of PJLME in B16F10 and A375 melanoma cells. Furthermore, Western blot experiments confirmed that the treatment of PJLME downregulates the expression of anti-apoptotic gene like Bcl2 and increase the expression profile of pro-apoptotic genes like Bax, Bad, and Bak in B16F10 melanoma cells. HUVEC (Human umbilical vein endothelial cells) tube formation assay clearly demonstrated the anti-angiogenic potential of PJLME. The study also revealed that PJLME has potential to inhibit the Akt and Erk signaling pathways which are participating in cancer cell proliferation, migration, invasion etc. The outcome of qRT-PCR and immunoblotting analysis clearly unveiled that PJLME treatment leads to downregulation of epithelial-mesenchymal transition (EMT) as well as stem cell markers. Finally, the in-vivo animal xenograft model study also revealed the anti-melanoma potential of PJLME by significantly inhibiting the B16F10 melanoma tumor growth in BALB/c mice model. The LC-ESI-MS/MS analysis of PJLME showed the presence of variety of bioactive molecules associated with anticancer effects. CONCLUSION: The outcome of the present investigation clearly demonstrated the anti-melanoma potential of PJLME against B16f10 melanoma cells. PJLME can be explored as an adjuvant or complementary therapy against melanoma cancer, however further studies are required to understand the clinical efficacy of PJLME. Nevertheless, it can be further explored as a promising resource for identification of novel anticancer candidate drug.

Identification of novel leads as potent inhibitors of HDAC3 using ligand-based pharmacophore modeling and MD simulation.

In the landscape of epigenetic regulation, histone deacetylase 3 (HDAC3) has emerged as a prominent therapeutic target for the design and development of candidate drugs against various types of cancers and other human disorders. Herein, we have performed ligand-based pharmacophore modeling, virtual screening, molecular docking, and MD simulations to design potent and selective inhibitors against HDAC3. The predicted best pharmacophore model 'Hypo 1' showed excellent correlation (R2 = 0.994), lowest RMSD (0.373), lowest total cost value (102.519), and highest cost difference (124.08). Hypo 1 consists of four salient pharmacophore features viz. one hydrogen bond acceptor (HBA), one ring aromatic (RA), and two hydrophobic (HYP). Hypo 1 was validated by Fischer's randomization with a 95% of confidence level and the external test set of 60 compounds with a good correlation coefficient (R2 = 0.970). The virtual screening of chemical databases, drug-like properties calculations followed by molecular docking resulted in identifying 22 representative hit compounds. Performed 50 ns of MD simulations on top three hits were retained the salient π-stacking, Zn2+ coordination, hydrogen bonding, and hydrophobic interactions with catalytic residues from the active site pocket of HDAC3. Total binding energy calculated by MM-PBSA showed that the Hit 1 and Hit 2 formed stable complexes with HDAC3 as compared to reference TSA. Further, the PLIP analysis showed a close resemblance between the salient pharmacophore features of Hypo 1 and the presence of molecular interactions in co-crystallized FDA-approved drugs. We conclude that the screened hit compounds may act as potent inhibitors of HDAC3 and further preclinical and clinical studies may pave the way for developing them as effective therapeutic agents for the treatment of different cancers and neurodegenerative disorders.